Project Summary/Abstract In 2016 there were 600,000 cancer-related deaths in the United States, with an estimated total cost of $130 billion. Given the aging population in the US, the incidence of cancer is expected to rise. Development of effective, widely applicable therapies for all cancers is of utmost importance. The Sondel group at the University of Wisconsin-Madison, with collaboration from many other talented researchers, has developed an immunotherapy that seeks to transform a tumor into a site of in situ vaccination. Using a combination of external beam radiation and a tumor-reactive fusion protein called an immunocytokine (an anti-tumor monoclonal antibody fused to the cytokine interleukin-2) injected into the tumor, our group has demonstrated effective recruitment of both innate and adaptive immune effector cells capable of clearing tumors in murine models of melanoma, neuroblastoma, and head and neck squamous cell carcinoma. These mice reject establishment of additional tumors, demonstrating memory response indicative of a vaccine effect. Despite a subset of treated mice experiencing complete response to this treatment, complete response is not universal. Preliminary studies of our in situ vaccine in mice with multiple tumors have shown that the presence of an untreated, distant tumor antagonizes development of an antitumor immune response at the treated tumor. This phenomenon, which we have termed ?concomitant immune tolerance (CIT),? is tumor- specific and is mediated, at least in part, by immune suppressive populations such as T regulatory cells. Importantly, preliminary work has established that additional low-dose external beam radiation delivered to distant tumor sites can reduce or eliminate CIT. We hypothesize that CIT is mediated by immune suppressive cells including Tregs derived from distant established tumor microenvironments, and that low-dose radiation can be used for immunomodulatory purposes to prime the system to be responsive to in situ vaccination. The goal of this proposal is to test this hypothesized mechanism for CIT, as well as develop a treatment approach that will overcome CIT and improve our in situ vaccine. This proposal will build on the expertise of the Sondel and Morris labs to probe the mechanism of CIT. In Aim 1, the impact of distant tumor size and location on the development of CIT will be determined. Immune changes in the tumor microenvironment of mice exhibiting CIT will be quantified, and connected to our hypothesized CIT mechanism. This will be accomplished using flow cytometry and ex vivo stimulation assays. Aim 2 seeks to establish a novel means of delivering low-dose molecular targeted radiotherapy to all sites of disease, with the goal of eliminating tumor-specific immune populations and generating a more potent systemic immune response to in situ vaccination. Achievement of these aims will provide insight into how these murine cancers avoid and escape immune destruction, how immune escape can be overcome in these settings, and will be used to establish a novel combination immunotherapy that could be readily translated into clinical testing.